The role of mass spectrometry for the structural characterization of peptides bound to Class I and Class II MHC molecules has been established. Sequence analysis of peptides in mixtures prepared from immunoaffinity purified proteins has been achieved by the combination of microcapillary HPLC with electrospray ionization/tandem mass spectrometry. The sensitivity and resolving power of the method is particularly suited for the identification of peptides that are recognized by individual T-cells. The sequences of 15 peptides eluted from HLA-A2.1 and HLA-B7 have been obtained. A sequence alignment has implicated two to three peptide positions important for specific interactions with the Class I molecule, and their importance was confirmed using synthetic peptides. Computer modeling was used to understand the specificity of peptide binding and to predict how defined changes in peptide structure would affect it. This technique is capable of accurately predicting the characteristics of peptide interactions, and it may be valuable for predictions involving other HLA molecules. Peptides derived from the processing of normal cellular proteins have been implicated in the formation of epitopes recognized by alloreactive T-cells and we have recently identified such a peptide as the epitope for a murine HLA-A2. 1 specific CTL. This same approach is expected to facilitate identification of peptides produced upon viral infection or cellular transformation. Peptides presented by Class II MHC molecules are 16-18 residues in length and exhibit ragged N- and C-termini. All of the peptides identified are derived from integral membrane proteins or secretory components. A large percent are derived from self Class I molecules and the Class II- associated invariant chain. The truncation at both the N- and C-termini indicated that the Class II molecules bind antigens from a pool of randomly generated peptides. Binding data on truncated peptides suggest that the peptide binding groove on Class II molecules is open at both ends. The predominance of peptides bound to Class II molecules that are derived from MHC - related proteins may be relevant to the etiology of autoimmune diseases linked to various alleles of Class II molecules. An investigation of the T-cell responses in autoimmune diseases should clarify the existing relationship between MHC restriction and disease pathogenesis.